Assessment of Cell Viability

Assessment of Cell Viability Simon Johnson, 1 Vy Nguyen, 1 and David Coder 2 UNIT 9.2 1 University of Washington School of Medicine, Seattle, Washington 2 Baybioscience, Irvine, California ABSTRACT Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes or the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre-fixation viability of fixed cells either before or after fixation with amine-reactive dyes suitable for a range of excitation wavelengths. Membrane-impermeable dead cell and live cell dyes as well as dye-exclusion procedures for microscopy are also included. Curr. Protoc. Cytom. 64:9.2.1-9.2.26. C 2013 by John Wiley & Sons, Inc. Keywords: cytometry flow cytometry imaging probes toxicology assessment of cell toxicity INTRODUCTION The method used to determine cell viability (and to a degree, the definition of viability) is often related to the phenomenon studied. Frequently, cell viability is thought of in somewhat negative terms. That is, one needs to exclude dead cells because they generate artifacts as a result of nonspecific binding and/or uptake of fluorescent probes. In addition to simple enumeration of live or dead cells present, there is a broad range of biologically relevant cytometric procedures that are related to the physiological state of the cells measured. For example, one may wish to measure cell morbidity in apoptosis, cell survival as a result of cytotoxicity, the potential of bacteria or microalgae to survive environmental insult and grow once normal conditions have been restored, or the potential of intracellular protozoa to undergo division cycles as part of the infective process. Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes or the uptake and retention of others. Here, methods are presented for staining nonviable cells by dye exclusion (indicative of an intact membrane) using the fluorescent, DNAbinding probes propidium iodide (PI; see Basic Protocol 1) and 7-amino actinomycin D (7-AAD; see Alternate Protocol 1). These probes may also be used in cells labeled with phycoerythrin (PE)-conjugated antibodies (see Alternate Protocol 2). The next two protocols present different aspects of physiological state that can be used to assess viability; one is based on esterase activity (see Alternate Protocol 3) and the other on mitochondrial membrane potential (see Alternate Protocol 4). For fixed cells, the state of viability prior to fixation can be determined using DNA-binding probes either before (see Alternate Protocols 5 and 6) or after (see Alternate Protocol 7) fixation. In addition, amine-reactive dyes (see Basic Protocols 2 and 3) can used with fixation and provide choices of excitation lasers. For assessment by microcopy, methods are provided that are based on the esterase activity of viable cells (see Basic Protocol 4) or simultaneous detection of live and dead cells (see Alternate Protocols 8 and 9). In contrast, the final method (see Alternate Protocol 10) is a dye-exclusion procedure for microscopy using trypan blue and a hemacytometer (APPENDIX 3A & 3B). Each of the above protocols requires a basic understanding of cell handling and flow cytometry/microscopy. Current Protocols in Cytometry 9.2.1-9.2.26, April 2013 Published online April 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142956.cy0902s64 Copyright C 2013 John Wiley & Sons, Inc. Studies of Cell Function 9.2.1

ASSESSMENT OF CELL VIABILITY USING PROBES FOR MEMBRANE INTEGRITY Live cells with intact membranes are distinguished by their ability to exclude dyes that easily penetrate dead or damaged cells. Staining of nonviable cells with propidium iodide (PI) has been performed on most cell types. Its broad application is most likely due to ease of use: the procedure is very simple, and the stained cells are bright red and easy to identify. Alternatively, the longer-wavelength emission of 7-amino actinomycin D (7-AAD) makes it easier to use simultaneously with phycoerythrin (PE) as a surface marker, as detailed in Alternate Protocol 2, or with fluorescein isothiocyanate (FITC). Although 7-AAD is not as bright as PI, permeable cells are easily distinguishable from live cells. BASIC PROTOCOL 1 Propidium Iodide Staining of Nonviable Cells Materials 2 mg/ml propidium iodide (PI) in PBS (store wrapped in foil 1 month at 4 C) Cell suspension PBS (APPENDIX 2A) 13 100 mm polystyrene culture tubes CAUTION: Propidium iodide is a suspected carcinogen and should be handled with care. In particular, be careful of particulate dust when weighing out the dye. Use gloves when handling it. 1. Add 1 μl of 2 mg/ml propidium iodide (2 μg/ml final) to approximately 10 6 washed cells suspended in 1 ml PBS in 13 100 mm polystyrene culture tubes. To use this procedure with cells that are also labeled with PE-conjugated antibodies, see Alternate Protocol 2. 2000 Number of cells 1500 1000 500 1 live 2 dead 0 1 10 100 1000 10,000 PI fluorescence Figure 9.2.1 Identification of nonviable cells with propidium iodide (PI). Nonviable cells are more than two decades brighter than the unstained, viable cells. Gating on a one-parameter histogram is sufficient to identify the viable population. Region 1: viable cells; region 2: nonviable cells. Assessment of Cell Viability 9.2.2 Current Protocols in Cytometry

2. Incubate 5 min in the dark on ice. 3. Analyze on flow cytometer with excitation at 488 nm and emission collected at >550 nm. PI is easily excited at 488 nm. The dye has a broad fluorescence emission and can be detected with photomultiplier tubes (PMTs) normally used for phycoerythrin ( 585 nm) or at longer wavelengths ( 650 nm). Amplify the PMT signal logarithmically to distinguish populations of permeable (and presumed dead) cells from viable cells. Adjust the PMT high voltage such that bright cells are well separated from dim, viable cells. It is often easy to identify nonviable cells on a bivariate plot of forward light scatter (see Fig. 9.2.1). Dead cells can be live-gated, but unless one is absolutely sure of the viable population, it is always much better to collect un-gated listmode data and perform gating after the raw data files are collected. Populations that may not be obvious on the flow cytometer display will be seen during subsequent data analysis. Moreover, any gating can be done and redone without losing cells. 7-AAD Staining of Nonviable Cells Additional Materials (also see Basic Protocol 1) 1 mg/ml 7-amino actinomycin D (7-AAD; see recipe) 1. Add 1 μl of 1 mg/ml 7-AAD (1 μg/ml final) to approximately 10 6 washed cells suspended in 1 ml PBS in 13 100 mm polystyrene culture tubes. To use this procedure with cells that are also labeled with PE-conjugated antibodies, see Alternate Protocol 2. 2. Incubate 30 min in the dark on ice. 3. Analyze on flow cytometer with excitation at 488 nm. Collect fluorescence emission with a 650-nm long-pass or a 670 ± 20 nm band-pass filter. 7-AAD is easily excited at 488 nm. The fluorescence emission of the dye has a peak at 670 nm. Use logarithmic amplification to distinguish permeable and bright cells from nonpermeable cells. Adjust the PMT high voltage to resolve a population of viable cells in the first decade of the 7-AAD fluorescence histogram as shown in region 1 of Figure 9.2.2A; nonviable cells are in region 2 of Figure 9.2.2A. ALTERNATE PROTOCOL 1 Use of PI or 7-AAD for Cells Labeled with PE-Conjugated Antibodies Additional Materials (also see Basic Protocol 1) PE-labeled cell suspension (UNIT 6.2) 1. Stain and incubate PE-labeled cells with PI (see Basic Protocol 1, steps 1 and 2) or 7-AAD (see Alternate Protocol 1, steps 1 and 2). 2. Analyze on flow cytometer with excitation at 488 nm. Use a 585 ± 20 nm band-pass filter for detection of PE fluorescence and a 650-nm long-pass filter for PI or 7-AAD. PI, 7-AAD, and PE are all excited by 488-nm light. Despite the separation by two detection filters, there is substantial overlap between the PE and PI or 7-AAD, requiring compensation between the two detectors. This problem is illustrated for PE and 7-AAD in Figure 9.2.2 (parts B and C), though the same procedure is used with PI. Note the typical discrimination of viable/nonviable cells. If one were to set a gate region on the viable cells in region 1 and use that for analysis in the absence of proper compensation, most PE-positive cells would be eliminated as nonviable (see Fig. 9.2.2B). ALTERNATE PROTOCOL 2 Studies of Cell Function 9.2.3 Current Protocols in Cytometry

A 180 160 140 Number of cells 120 100 80 60 1 live 40 20 2 dead B PE fluorescence 10,000 0 1 10 100 1000 10,000 7-AAD fluorescence 1 2 1 2 live live 1000 100 10 C 1 3 4 1 10 100 1000 10,000 7-AAD fluorescence 3 4 1 10 100 1000 10,000 7-AAD fluorescence Figure 9.2.2 Effects of gating and compensation with 7-AAD. (A) Gating discriminates live cells. One-parameter histogram of logarithmically amplified 7-AAD fluorescence using a 650-nm longpass filter. Mouse spleen cells are labeled only with 7-AAD. Note the peak of dead cell population in region 2 at a relative brightness between 100 and 200 (about ten-fold dimmer than what is expected for propidium iodide). The live cell population that occupies the first decade in the histogram (region 1) is 7-AAD negative and constitutes the majority of the cells in the population. (B) Uncompensated phycoerythrin fluorescence in the presence of 7-AAD. A bivariate plot of mouse spleen cells labeled with 7-AAD and a PE-labeled antibody to a cell surface antigen. Note the two small populations of dead cells in regions 2 and 4 and the large population of live cells that occupies region 2. If gating was performed before adequate compensation was achieved, then most of the viable PE-positive cells could be lost. (C) Compensation of PE with 7-AAD. Distribution of cells from same sample as in B. Note the dead cells are in the same location as in B, but the live cells are now clearly resolved from 7-AAD positive populations. At this point, the live cell gate defined in region 2 of A is valid. Using a bivariate histogram of log 7-AAD versus log PE fluorescence, adjust the PE- 7-AAD compensation such that PE-positive, 7-AAD-negative cells are above the PEnegative, 7-AAD-negative population (see Fig. 9.2.2C). Once proper compensation of the long-wavelength component of PE is subtracted from the output of the detector for 7-AAD, the problem disappears. Assessment of Cell Viability 9.2.4 Current Protocols in Cytometry

ASSESSMENT OF CELL VIABILITY USING PROBES OF PHYSIOLOGICAL STATE These protocols describe the use of probes that require a specific cellular function in addition to an intact membrane. Alternate Protocol 3 describes the use of fluorescein diacetate (FDA), which requires cellular esterase activity, and Alternate Protocol 4 describes the use of rhodamine 123 as a probe for mitochondrial membrane potential. Fluorescein Diacetate Staining of Viable Cells Cell viability can be assessed directly through the presence of cytoplasmic esterases that cleave moieties from a lipid-soluble nonfluorescent probe to yield a fluorescent product. The product is charged, and thus is retained within the cell if membrane function is intact. Hence, viable cells are bright and nonviable cells are dim or nonfluorescent. Typical probes include fluorescein diacetate (FDA, described here), carboxyfluorescein, and calcein. Variations in uptake or retention of the dye among individual cells or under different conditions affect the efficacy of particular probes. ALTERNATE PROTOCOL 3 Additional Materials (also see Basic Protocol 1) 1 mg/ml fluorescein diacetate (FDA; prepare fresh in acetone in a 13-mm glass culture tube and cover with foil) Cell suspension in culture medium appropriate for the cell type 1. Add 2 μl of 1 mg/ml FDA (2 μg/ml final) to approximately 10 6 cells in 1 ml medium in a 13 100 mm polystyrene culture tube. 2. Vortex to mix and incubate 15 min at 37 C. 3. Analyze on flow cytometer immediately with excitation at 488 nm. Collect fluorescence using a 530 ± 20 nm band-pass filter. FDA is excited by 488-nm light and fluoresces green. Filters used for measuring fluorescein (e.g., 530 ± 20 nm band-pass) are sufficient to visualize the nonfluorescent cells on the same scale. Use logarithmic amplification of the PMT output. Cells that take up and retain free fluorescein are very bright (approximately two decades brighter on a logarithmic scale) and should be easily distinguishable from nonfluorescent, nonviable cells. Unless one is absolutely sure about the location of viable cells in the histograms, it is preferable to collect listmode data files of all samples and perform gating after the raw data files are collected, to avoid the danger of inadvertent loss of viable cells. Rhodamine 123 Staining of Viable Cells Another property of viable cells is the maintenance of electrochemical gradients across the plasma membrane. Functional subsets of this general phenomenon include the maintenance of ph and other ion gradients as well as the capacity for energy-yielding metabolism in mitochondria. These physiological processes can be exploited to distinguish viable from nonviable cells. One of the most commonly used probes for identifying viable cells is rhodamine 123, a cationic lipophilic dye that partitions into the low electrochemical potential of mitochondrial membranes. Active mitochondria in viable cells are stained bright green; loss of gradients within nonviable cells results in loss of the dye. Additional Materials (also see Basic Protocol 1) 1 mg/ml rhodamine 123 (prepare fresh in distilled water) Cell suspension in culture medium appropriate for the cell type 1. Add 5 μl of 1 mg/ml rhodamine 123 (5 μg/ml final) to approximately 10 6 cells in 1 ml medium in a 13 100 mm polystyrene culture tube. 2. Vortex to mix and incubate 5 min at 37 C; return to room temperature. ALTERNATE PROTOCOL 4 Studies of Cell Function 9.2.5 Current Protocols in Cytometry

3. Analyze immediately with excitation at 488 nm. Collect fluorescence using a 530 ± 20 nm bandpass filter. Rhodamine 123 absorbs 488-nm light and fluoresces green. Collect fluorescence through a filter that transmits at 530 nm, as for FITC. PMT output should be amplified logarithmically. Viable cells are brighter than nonviable cells, though with some cell types there may be some overlap (see Fig. 9.2.3A). A bivariate plot of forward scatter versus rhodamine 123 fluorescence can be useful to distinguish viable from nonviable cells (see Fig. 9.2.3B). Unless one is absolutely sure about the location of viable cells in the histograms, it is preferable to collect listmode data files of all samples and perform gating after the raw data are collected, to avoid the danger of inadvertent loss of viable cells. A 3000 2500 Number of cells 2000 1500 1000 ungated live gate 500 B 0 10,000 1 10 100 1000 10,000 Rhodamine 123 fluorescence Rhodamine 123 fluorescence 1000 100 10 live 1 0 dead 50 100 150 200 250 Forward light scatter Assessment of Cell Viability 9.2.6 Figure 9.2.3 Effects of gating with rhodamine 123. (A) Identification of live cells after gating. Rhodamine 123 may not always completely resolve viable from nonviable cells as indicated in the un-gated histogram (dotted line). Gating on forward light scatter versus rhodamine 123 fluorescence helps separate both populations. Note the histogram of the gated population of viable cells (solid line) overlaid on the ungated population. (B) A bivariate plot of forward light scatter versus rhodamine 123 fluorescence helps to resolve live (rhodamine 123 bright) and dead (rhodamine 123 dim) populations. Debris is gated out at the same time. Current Protocols in Cytometry

ASSESSMENT OF CELL VIABILITY IN FIXED CELLS For reasons of safety or convenience, it is frequently necessary to fix cells prior to analysis. Data analysis is less ambiguous if nonviable or damaged cells can be eliminated, but the methods discussed above will not work, because fixation will render all cells permeable. There are, however, DNA probes that penetrate and stain dead or damaged cells and that can withstand fixation. Ethidium monoazide (EMA) is positively charged and penetrates the membranes of dead or damaged cells but not live ones. EMA can be photochemically cross-linked with short exposure to visible light; after the excess dye is washed away, the cells are fixed. Another DNA-binding fluorochrome used for staining nonviable cells is laser dye styryl-751 (LDS-751). The procedure is somewhat more simple, as staining is done after fixation and does not require cross-linking. Recent advances in probe design have yielded live cell exclusion dyes of multiple spectral and chemical varieties, allowing for greater flexibility in experimental design. Among these newer amine-reactive dyes are the Live/Dead probes (Invitrogen/Life Technologies), Horizon Fixable Viability Stain 450 (Becton Dickinson), and the Fixable Viability Dye efluor dyes (ebioscience). These probes adhere to intact membranes of live cells, resulting in weak staining, and diffuse into cells with compromised cell membranes, resulting in bright staining easily distinguishable from dead or unstained cells. The available probes provide a range of options in their excitation/emission properties. In addition, these probes are compatible with aldehyde-based fixation, allowing for viability analysis of fixed or non-fixed cells if staining is performed prior to fixation. Carboxymethylfluorescein diacetate (Cell Tracker CMFDA, Invitrogen/Life Technologies) provides an alternative to the cell-exclusion dyes that is still reliant on membrane integrity. This dye freely enters cells and reacts with intracellular esterases to form a nondiffusible product. Cells with intact membranes thus retain the fluorescent probe while the probe diffuses from damaged cells. Positive staining thus identifies live cells while weak or absent staining designates damaged or dead cells. The brightness of the dye allow for easy distinction of these populations of cells. As with the Live/Dead fixable dyes, the Cell Tracker dyes are compatible with aldehyde-based fixation and are available in multiple spectral variants. Live/Dead Far Red Fixable Staining of Cells for Assessment of Viability Materials Live/Dead Far Red Dead stock solution (see recipe) Cell suspension, washed Phosphate-buffered saline (PBS; APPENDIX 2A) 100 mm digitonin (see recipe; optional/recommended). 37% formaldehyde (Sigma, cat. no. 252549; optional) PBS (APPENDIX 2A) with 1% (w/v) bovine serum albumin (BSA) or cell culture medium without phenol red supplemented with 1% BSA 13 100 mm culture tubes or any suitable container for delivering cells to the flow cytometer for analysis Tabletop centrifuge Flow cytometer 1. Add 1 μl of Far Red Dead fixable cell stain stock to approximately 10 6 washed cells suspended in 1 ml PBS in 13 100 mm polystyrene culture tubes or alternate container. BASIC PROTOCOL 2 Studies of Cell Function 9.2.7 Current Protocols in Cytometry

If multiple cell samples will be compared prepare the Far Red Dead staining solution prior to resuspending cells in PBS by diluting the dye in PBS at a 1:1000 ratio, preparing 1 ml of solution per sample. 2. Optional (recommended): Prepare a positive control for dead cells by adding 1 μl of 100 mm digitonin to one 1-ml aliquot of cells containing approximately 10 6 cells and mixing by gently pipetting up and down. 3. Incubate protected from light for 20 to 30 min at room temperature or on ice. 4. Optional: Fix cells by adding 100 μl of 37% formaldehyde and incubating for 15 min at room temperature. 5. Wash cells by centrifuging 5 min at 700 to 1000 g, room temperature, in a tabletop centrifuge, removing the supernatant by gentle pipetting, and resuspending in PBS with 1% (w/v) BSA or cell culture medium with 1% (w/v) BSA. 6. Resuspend cells in PBS with 1% (w/v) BSA or cell culture medium with 1% (w/v) BSA. Store, protected from light, in polystyrene culture tubes on ice. 7. Analyze on flow cytometer with excitation at 635 nm. Collect fluorescence emission with a 650-nm long-pass or a 670 ± 20 nm band-pass filter. A 55 live cell sample B 52 dead cell control (100 M digitonin) 41 17% 39 97% count 28 count 26 14 13 C 0 10 0 10 1 10 2 10 3 10 4 660-720 (Far Red Dead Fixable Cell Stain) 81 overlay 660-720 (Far Red Dead Fixable Cell Stain) 61 live cell sample count 41 20 10 M digitonin dead cell control (100 M digitonin) 0 10 0 10 1 10 2 10 3 10 4 660-720 (Far Red Dead Fixable Cell Stain) Figure 9.2.4 Example of staining and flow cytometry analysis of live/dead populations using fixed cells stained that had been stained with Live/Dead Deep Red. (A) Sample plot of a confluent population of cells. Live cells stain weakly, while dead cells, in this case representing 17% of the culture population, stain brightly. (B) A positive control for dead cells produced by treating cells with 100 μm digitonin during staining. (C) An overlay of the sample, dead cell control, and an intermediate concentration of digitonin demonstrating that partial staining may occur if cells are partially permeabilized. This was only observed when cells were permeabilized using digitonin and does not occur in normal cell populations. Assessment of Cell Viability 9.2.8 Current Protocols in Cytometry

Far Red Dead fixable cell stain has an excitation peak at 650 nm and is easily excited at 633/655 nm. The bright staining of dead/permeable cells is easily distinguished from the weak staining of impermeable cells (see Fig. 9.2.4). A positive control for dead cells is recommended and can be used to aid in setting an optimal cytometer gain and providing a population on which to set a dead-gate if desired. 100 μm digitonin added to a cell aliquot during the staining step provides a simple positive control for the bright staining of dead cells. Lightly permeabilized cells may stain with an intensity intermediate to live or dead cells (see Fig. 9.2.4C). These cells were not observed under normal culture conditions but were found to be a product of low-dose digitonin treatment. As with all live/dead dyes, viability should be carefully defined as part of the experimental design prior to running any samples, and suitable positive and negative controls should be included to allow for unambiguous distinction of defined viable and nonviable populations. Live/Dead fixable dyes are available in a number of spectral variations, providing flexibility in cytometer choice, wavelength combinations, and experimental design. Violet-Excited Fixable Staining of Cells for Assessment of Viability Materials Phosphate-buffered saline (PBS; APPENDIX 2A), azide-free and serum/protein-free Cell suspension ( 1 10 6 cells/ml) 100 mm digitonin (see recipe; optional/recommended). Fixable Viability Dye efluor 506 (ebioscience; supplied as a pre-diluted solution in anhydrous DMSO; protect from light and moisture; store at 70 C with desiccant; may be freeze-thawed up to 20 times) 37% formaldehyde (Sigma, cat. no. 252549; optional) PBS (APPENDIX 2A) with 1% (w/v) bovine serum albumin (BSA) or cell culture medium without phenol red supplemented with 1% BSA Culture tubes (13 100 mm) or any alternative suitable container for delivering cells to the flow cytometer for analysis Flow cytometer 1. Place 1 ml of an 1 10 6 cell/ml suspension in a 13 100 mm polystyrene culture tube, or equivalent container. Wash cells twice in azide-free and serum/protein-free PBS, each time by centrifuging 5 min at 1000 g, room temperature. Remove the supernatant from the final wash. 2. Optional/recommended: Prepare a positive control for dead cells by adding 1 μl of 100 mm digitonin to one 1-ml aliquot of 1 10 6 cells and mixing by gentle pipetting. A dead control is recommended if the percentage of dead cells is expected to be less than 5%. 3. Resuspend cells at 1 10 10 6 /ml in azide-free and serum/protein-free PBS. 4. Add 1 μl of efluor 506 fixable dye per 1 ml of cells and vortex immediately. Allow vial to equilibrate to room temperature before opening. 5. Incubate for 30 min at to 8 C; protect from light. 6. Wash cells one to two times with PBS. 7. Optional: Fix cells by adding 100 μl of 37% formaldehyde and incubating for 15 min at room temperature. BASIC PROTOCOL 3 Studies of Cell Function 9.2.9 Current Protocols in Cytometry

8. Wash cells once in PBS with 1% BSA or cell culture medium with 1% BSA, each time by centrifuging 5 min at 700 to 1000 g, room temperature. Remove the supernatant from the final wash. 9. Resuspend cells in PBS with 1% BSA or cell culture medium with 1% BSA. Store, protected from light on ice. 10. Analyze on flow cytometer with excitation at 405 nm. Collect fluorescence emission with a 510 ± 50-nm band-pass filter. Staining with efluor 506 fixable dye must be done in azide-free and serum/protein-free PBS. For consistent staining of cells, do not stain cells in less volume than 0.5 ml. Cells may be stained with efluor 506 fixable dye before or after surface staining. After staining with efluor 506 fixable dye, cells may also be cryopreserved for analysis at a later time. The dye is titrated for use with mouse thymocytes. It is recommended that you titrate the dye concentration for use with your cells. A range of fixable efluor dyes is available with excitation from violet to red. ALTERNATE PROTOCOL 5 Carboxymethylfluorescein Diacetate (Cell Tracker CMFDA) Staining of Cells for Assessment of Viability by Flow Cytometry Additional Materials (also see Basic Protocol 1) 5 mm carboxymethylfluorescein diacetate (see recipe) 1. Add 1 μl of 5 mm CMFDA (5 μm final) to approximately 10 6 washed cells suspended in 1 ml PBS in 13 100 mm polystyrene culture tubes. Recommended: include one unstained sample to provide an unstained control (see Fig. 9.2.5). 2. Incubate 15 min in the dark at room temperature. 3. Wash once in PBS (centrifuging 5 min at 700 to 1000 g, room temperature) and incubate 5 min in the dark at room temperature to destain. A 74 carboxymethyl fluorescein diacetate (Cell Tracker CMFDA) staining analyzed by flow cytometry 56 98.83 98.91 86.22 count 37 19 0 10 0 10 1 10 2 525-530 (Cell Tracker Green) 10 3 10 4 unstained cells dead cell control (0.05% Tx-100) live cell sample Assessment of Cell Viability 9.2.10 Figure 9.2.5 Sample staining using Cell Tracker CMFDA. Stained dead cells are brighter than unstained cells while stained live cells are >100 fold brighter than dead cells and easily distinguished from the dead population. Current Protocols in Cytometry

4. Optional: Fix cells by adding 100 μl of 37% formaldehyde and incubating for 15 min at room temperature. 5. Resuspend cells in PBS with 1% BSA or cell culture medium with 1% BSA. Store, protected from light, in polystyrene culture tubes on ice. 6. Analyze on flow cytometer with excitation at 488 nm. Collect fluorescence emission using a filter appropriate for 520 nm emission. CMFDA is a very bright dye and live cells are easily distinguished from dead cells. Ethidium Monoazide Staining of Nonviable Cells Prior to Fixation Additional Materials (also see Basic Protocol 1) 50 μg/ml ethidium monoazide (EMA; see recipe) 1% (w/v) paraformaldehyde in PBS (see APPENDIX 2A for PBS; store mixture 1 week at 4 C and discard if precipitate forms) 40-W fluorescent light 1. Add 10 μl of50μg/ml EMA ( 5 μg/ml final) to approximately 10 6 washed cells suspended in 100 μl PBS in a 13 100 mm polystyrene culture tube. Preparations of EMA dye vary. The exact concentration needed may range from 1 to 5 μg/ml. 2. Place tubes on ice 18 cm beneath a 40-W fluorescent light for 10 min. EMA diffuses into dead cells and intercalates into DNA. When exposed to light, EMA is then covalently bound to the DNA 3. Wash and fix cells by adding 1 ml μl of 1% paraformaldehyde to the cell pellet. Incubate 1 hr at room temperature. 4. Analyze on flow cytometer with excitation at 488 nm. Collect fluorescence emission using a 630-nm long-pass filter; amplify PMT output logarithmically. EMA excites with 488-nm light and fluoresces well into the red region of the spectrum. EMA does not fluoresce as brightly as propidium iodide, so discrimination of nonviable, EMA-bright cells from viable cells may be less obvious. A bivariate plot of forward light scatter versus EMA fluorescence may aid in distinguishing viable (EMA-negative) cells. Dead cells can be live-gated, but unless one is absolutely sure of the viable population, it is always better to collect un-gated listmode data and perform gating after the raw data files are collected. Populations that may not be obvious on the flow cytometer display will be seen during subsequent data analysis. Moreover, any gating can be done and redone without losing cells. LDS-751 Staining of Previously Nonviable Cells After Fixation Additional Materials (also see Basic Protocol 1) 1% (w/v) paraformaldehyde in PBS (see APPENDIX 2A for PBS; store mixture 1 week at 4 C and discard if precipitate forms) 2 μg/ml LDS-751 (laser dye styryl-751) working solution (see recipe) 1. Wash approximately 10 6 cells in PBS in a 13 100 mm polystyrene culture tube. 2. Fix cells by adding 1 ml μl of 1% paraformaldehyde to the cell pellet. Incubate 1 hr at room temperature. 3. Add 10 μlof2μg/ml LDS-751 working solution to 1 ml fixed cells at a concentration of approximately 10 6 cells per ml. ALTERNATE PROTOCOL 6 ALTERNATE PROTOCOL 7 Studies of Cell Function 9.2.11 Current Protocols in Cytometry

10 0 10 1 10 2 10 3 10 4 efluor 506-H 0 1000 2000 FSC-H 3000 4000 Figure 9.2.6 Identification of dead cells with efluor 506. The bright dead population is clearly distinguished from unstained cells. While the excitation was with 405-nm light, the detection filter, (530/30), was slightly suboptimal for detection. 4. Incubate overnight at room temperature in the dark. 5. Analyze on flow cytometer with excitation at 488 nm. Collect fluorescence emission using a 650-nm long-pass filter. LDS-751 is excited with 488-nm light and emits in the red portion of the spectrum; the 650-nm long-pass filter is adequate to separate red fluorescence from other fluorochromes and scattered laser light. Amplify the PMT output logarithmically to distinguish bright, nonviable cells from dim, viable cells and from nonfluorescent red cells or debris. A bivariate plot of forward scatter versus log LDS-751 fluorescence can help identify populations. Dead cells can be live-gated, but unless one is absolutely sure of the viable population, it is always much better to collect un-gated listmode data and perform gating after the raw data files are collected. Populations that may not be obvious on the flow cytometer display will be seen during subsequent data analysis. Moreover, any gating can be done and redone without losing cells. ASSESSMENT OF CELL VIABILITY BY MICROSCOPY Determination of cellular viability by microscopy provides data complementary to that obtained using flow cytometry analysis but also allows for simultaneous examination of cell morphology, examination of subcellular structures using fluorescent proteins or vital dyes, or, when fixable viability dyes are used, analysis of viability at the time of fixation combined with immunologically based staining. Microscopy may also be the preferred method of viability assessment in cells that are not easily amenable to flow cytometry analysis due to culture conditions or cellular properties. Analysis of viability by fluorescence microscopy has become increasingly versatile in recent years, with the development of many spectral alternatives in both fixable and nonfixable dyes. Determining viability of a population of cells in culture is relatively easy, but usually requires the addition of a viability-independent cell stain. Flow cytometry may be the preferred method for viability determination in some cases, given that each cell is recorded as an event and a viability-independent counterstain is not necessary. Assessment of Cell Viability 9.2.12 Carboxymethylfluorescein diacetate (Cell Tracker CMFDA), described in Basic Protocol 1, is a bright, cell membrane integrity sensitive dye that provides a clear distinction between intact and damaged or dead cells (Fig. 9.2.7). CMFDA is easily combined with Hoechst 33342 to provide a cell count with which to determine percent viability. Current Protocols in Cytometry

A 100 Carboxymethylfluorescein diacetate (Cell Tracker CMFDA) staining of Triton X-100-treated cells B Concentration Triton X-100 (percent) 0 0.05 90 Percent stained 80 70 60 50 40 30 20 10 CMFDA Hoechst 33342 0 0.0001 0.001 0.01 Concentration of Triton X-100 (percent) 0.1 Figure 9.2.7 Example of viability analysis using Cell Tracker CMFDA and microscopy. (A) Cells are efficiently permeabilized using Triton X-100 as a positive control and CMFDA shows a steep dose response curve indicating high specificity for permeabilized cells. (B) Live cells are extremely bright and easily distinguished from dead cells. A counterstain is necessary to present data as a percent of total. CMFDA is also fixable and alternative spectral variants are available (including CMAC, see Alternate Protocol 2), though each spectral variant is likely to have a unique sensitivity to membrane integrity. Combined use of membrane-impermeable dead cell dyes (those that only enter damaged or dead cells) and live cell dyes (those that are retained in living cells but diffuse out of damaged or dead cells) allows for detection of viability using multiple measures simultaneously. These combinations allow for a more specific definition of viability in situations where viability status may be ambiguous using only one dye. Calcein AM and EtHD-1 (Alternate Protocol 1) make one such dye combination. Figure 9.2.8 demonstrates the differences in sensitivity of these dyes, with low-dose Triton X-100 producing cells that do not accumulate either Calcein AM or EtHD-1, thus staining neither viable nor nonviable cells. A stringent viability cutoff could designate only cells that are both negative for EtHD-1 and positive for Calcein AM as viable. Sytox AADvanced (a derivative of 7-AAD) and chloromethylaminocoumarin (Cell Tracker Blue CMAC) provide an alternative combined set of live/dead dyes. Sytox and Cell Tracker are both available in multiple spectral variants, allowing for selection of dyes compatible with the available microscopy equipment. Cell Tracker dyes are fixable, as discussed, allowing for fixation post-imaging and further analysis. Finally, assessment of cell viability may be accomplished with a simple microscope, using dyes that mark nonviable cells by dye exclusion. The most commonly used dye is trypan blue, but others may be used as well; see Background Information for details on other useful stains. Viable cells have intact membranes and exclude the dye; nonviable cells are labeled with the dye and are visible with brightfield optics. As well as being useful as a means of assessing functional integrity, trypan blue exclusion is widely used as an objective method of determining viable cell count prior to using cells; a simple protocol for this application is presented, along with other basic cell culture techniques, in APPENDIX 3B. Studies of Cell Function 9.2.13 Current Protocols in Cytometry

Percent stained A 100 90 80 70 60 50 40 30 20 10 Calcein AM and EthD-1 staining of Triton X-100 treated cells Calcein AM (viable) EthD-1 (inviable) 0.01% Triton X-100 0 0.001 0.01 Concentration of Triton X-100 (percent) 0.1 B EthD-1 Calcein AM Hoechst 33342 Concentration Triton X-100 (percent) 0 0.01 0.05 Figure 9.2.8 Example of dual staining with Calcein AM and EthD-1. (A) Analysis of dual-stained cells permeabilized with Triton X-100 demonstrates that while both dyes are highly specific for their target cells (they have steep drop-offs of staining in the dose response curve), they are slightly non-overlapping in their staining. Calcein AM is lost before EthD-1 stains cells (see inlay). This analysis did not include weakly staining cells, and adjustment of brightness to include these cells may reduce the observed lack of complementation. (B) Cells stained with either dye are extremely bright and easy to distinguish from unstained cells, although intermediate permeabilization can result in a lack of either stain. BASIC PROTOCOL 4 Assessment of Cell Viability 9.2.14 Carboxymethyl Fluorescein Diacetate (Cell Tracker CMFDA) Staining of Cells for Assessment of Viability by Microscopy Materials 5 mm carboxymethylfluorescein diacetate (see recipe) 1 mm Hoechst 33342 (Sigma) in phosphate-buffered saline (PBS; APPENDIX 2A) Cell culture medium (prewarmed to 37 C) Cultured cells on chambered coverglass (Nunc, cat. no. 155379) or grown on coverslips (for fixed only) Phosphate-buffered saline (PBS; APPENDIX 2A) Fluorescence microscope with filters appropriate for FITC and DAPI 1. Add 1 μl of 5 mm CMFDA (5 μm final) and 10 μl of 1 mm Hoechst 33342 (10 μm final) in PBS per ml to pre-warmed cell culture medium, making enough to cover cells completely (1 ml per well in covered chamber slides). Do not add Hoechst if fixing cells (optional, step 4). 2. Aspirate medium from cells on chambered coverglass and replace with the medium containing dyes from step 1. 3. Incubate 15 min in the dark under cell culture conditions. 4. Wash once with warm PBS, replace medium, and incubate 10 to 15 min in the dark at cell culture conditions to destain. 5. Optional: Fix cells by adding 100 μl of 37% formaldehyde per ml medium, gently mixing, and incubating for 15 min at room temperature. Wash once with PBS and store at 4 CinPBS. 6. Image cells by microscopy using filters appropriate for FITC (for CMFDA) and DAPI (for Hoechst). Current Protocols in Cytometry

If cells were fixed using optional step 4 DNA can be stained using any compatible DNA fluorophore, such as DAPI, to provide a counterstain. CMFDA is a very bright dye and live cells are easily distinguished from dead cells (see Fig. 9.2.7). If fixed, cells can be used for additional staining. Simultaneous Detection of Live and Dead Cells by Microscopy using Calcein AM, EthD-1, and Hoechst 33342 Materials 2 mm Calcein AM (see recipe) 4 mm ethidium homodimer 1 (EtHD-1; see recipe) Cell culture medium (prewarmed to 37 C) Cultured cells on chambered covers glass (Nunc, cat. no. 155379) or grown on coverslips (for fixed cells only) 100 mm digitonin (see recipe) or 50% (v/v) Triton X-100 in H 2 O; optional/recommended Phosphate-buffered saline (PBS; APPENDIX 2A) Fluorescence microscope with filters for FITC (for Calcein AM), Texas Red (for EthD-1), and DAPI (for Hoechst) 1. Add 1 μl of 2 mm Calcein AM (2 μm final), 1 μl of 4 mm EtHD-1 (1 μm final), and 10 μl of 1 mm Hoechst 33342 (10 μm final) in PBS per ml to prewarmed cell culture medium, making enough to cover the cells completely (1 ml per well in covered chamber slides). 2. Aspirate medium from the cells and replace with medium containing dyes. 3. Optional (recommended): Add 1 μl of 100 mm digitonin in DMSO per ml medium, or add Triton X-100 to 0.05% (v/v; final concentration) to cells to create a positive control. 4. Incubate 15 min in the dark under cell culture conditions. 5. Wash once with warm PBS, replace with fresh medium, and incubate 10 to 15 min in the dark under cell culture conditions to destain. 6. Image cells by microscopy using filters appropriate for FITC (for Calcein AM), Texas Red (for EthD-1), and DAPI (for Hoechst). Calcein AM and EthD-1 are both very bright, very sensitive dyes, but loss of Calcein AM staining can be observed in permeabilized cells, indicating that defining loss of viability may depend on the dye analyzed. Because of the slight lack of overlap between the two dyes, a nuclear dye should always be included so that the percent viable or nonviable cells is reflective of the total cell count, rather than the two fluorophores alone. Furthermore, viability should be carefully defined prior to the start of the experiment, such that it is predetermined whether cells lacking either stain are considered viable or not (see Fig. 9.2.8). Triton X-100 provides a simple and cheap positive control but can, at high concentrations, lift cells from the surface of the plate and prevent imaging. Digitonin, on the other hand, is more specific for membrane permeabilization but is toxic and expensive. Triton should be prepared as a 50% stock prior to use to allow for pipetting of the viscous compound. ALTERNATE PROTOCOL 8 Simultaneous Detection of Live and Dead Cells by Microscopy using Chloromethylaminocoumarin (Cell Tracker Blue CMAC) and Sytox AADvanced Materials 1 mm CMAC (see recipe) 1 mm Sytox AADvanced (see recipe) ALTERNATE PROTOCOL 9 Studies of Cell Function 9.2.15 Current Protocols in Cytometry

Cell culture medium (prewarmed to 37 C) Cultured cells on chambered covers glass (Nunc, cat. no. 155379) or grown on coverslips (for fixed cells only) 100 mm digitonin (see recipe) or 50% (v/v) Triton X-100 in H 2 O; optional/recommended Phosphate-buffered saline (PBS; APPENDIX 2A) Fluorescence microscope with filters appropriate for 353 nm absorption/466 nm emission (CMAC) and 546 nm absorption/647 emission (for Sytox AADvanced) 1. Add 1 μl of1mmcmac(1μm final) and 1 μl of 1 mm Sytox AADvanced to prewarmed cell culture medium, making enough to cover cells completely (1 ml per well in covered chamber slides). 2. Aspirate medium and replace with medium containing dyes. 3. Optional (recommended): Add 1 μl of 100 mm digitonin in DMSO per ml medium or Triton X-100 to 0.05% (v/v; final concentration) to cells to create a positive control. 4. Incubate 15 min in the dark under cell culture conditions. 5. Wash once with warm PBS, replace with fresh medium, and incubate 10 to 15 min in the dark under cell culture conditions to destain. 6. Image cells by microscopy using filters appropriate for 353 nm absorption/466 nm emission (CMAC) and 546 nm absorption/647 emission (for Sytox AADvanced). Calcein AM and EthD-1 are both very bright, very sensitive dyes. These dyes are highly complementary in their staining (see Fig. 9.2.9) with cells generally staining positively for one of the two dyes. A spectrally compatible nuclear dye may be included to provide a total cell count, as in the previous protocols. This protocol can be easily adapted depending on the spectral requirements of the experiment. The variety of Cell Tracker and Sytox probes available provides flexibility in dye combinations and allows for selection of dyes that are spectrally compatible with additional probes. A 100 Chloromethylaminocoumarin (Cell Tracker Blue CMAC) and Sytox AADvanced staining of Triton X-100 treated cells B Concentration Triton X-100 (percent) 0 0.025 90 Percent stained 80 70 60 50 40 30 20 10 Cell Tracker Blue (viable) Sytox AADvanced (inviable) 0 0.0001 0.001 0.01 Concentration of Triton X-100 (percent) 0.1 CMAC Sytox AADvanced Figure 9.2.9 Sample staining using Cell Tracker Blue CMAC and Sytox AADvanced. (A) Dose response curve for staining and permeabilization with Sytox AADvanced and CMAC. (B) These dyes appeared to be highly complementary, with no apparent intermediate dose where cells lacked staining for either dye, although no total cell staining dye was included in this analysis. 9.2.16 Current Protocols in Cytometry

Trypan Blue Staining Additional Materials (also see Basic Protocol 1) 0.4% (w/v) trypan blue in PBS (store up to 1 year at room temperature in the dark; filter if a precipitate forms; for PBS, see APPENDIX 2A) Serum-free culture medium (APPENDIX 3B optional) Additional materials for cell counting with a hemacytometer (APPENDIX 3A) 1. Add an equal volume of 0.4% trypan blue to a cell suspension at a concentration of approximately 10 6 cells per ml. Use PBS or a serum-free medium for the cell suspension. Serum proteins may stain with trypan blue, resulting in falsely depressed viable counts. 2. Incubate at room temperature for 3 min and load into a hemacytometer. Using brightfield optics, count cells in three separate fields (see APPENDIX 3A for use of hemacytometer). Count nonviable, deep blue cells as well as viable, clear cells. 3. Calculate viability: % viable = (number viable cells/number total cells) 100. ALTERNATE PROTOCOL 10 REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX. 7-AAD (7-amino actinomycin D), 1.0 mg/ml Dissolve 1.0 mg 7-AAD in 50 μl of dimethyl sulfoxide (DMSO), and then add 950 μl ofpbs(appendix 2A). Store 1 month in the dark at 4 C. As 7-AAD is not water soluble, an organic solvent is required. Although the mutagenicity of 7-AAD is unknown, caution should be exercised when handling the dye. Calcein AM (calcein acetoxymethyl ester), 2 mm Spin down a tube of 50 μg desiccated Calcein AM powder (Invitrogen, cat. no. C3100MP) to collect dye at the bottom of the tube. Dissolve in 5 μl of DMSO. Spin again to ensure that all powder is collected at bottom of tube. Store protected from light and moisture up to 1 year at 20 C. Cell Tracker Blue CMAC (chloromethylaminocoumarin), 1 mm Dissolve 5 mg of chloromethylaminocoumarin (Invitrogen, cat. no. C2110) in 238.5 μl of DMSO. Aliquot and store protected from light and moisture at 20 C. CMFDA (carboxymethylfluorescein diacetate), 5 mm Dissolve 1 mg of CMFDA (Invitrogen/Life Technologies, cat. no. C2925) in 420 μl of sterile DMSO. Aliquot and store protected from light and moisture at 20 C. Digitonin, 100 mm Spin down a tube of 100 mg desiccated digitonin (Sigma-Aldrich, cat. no. D141-100MG). Add 813.5 μl DMSO and dissolve by gently pipetting up and down. Store at 20 C. EMA (ethidium monoazide), 50 μg/ml Dissolve ethidium monoazide at 5 mg/ml in PBS (APPENDIX 2A). Dilute to 50 μg/ml (working strength) and divide into 0.5-ml aliquots. Wrap in aluminum foil and store 6 months at 20 C. Thaw immediately before use. Discard unused workingstrength dye. EMA is very light sensitive; keep wrapped in foil or in the dark. Studies of Cell Function 9.2.17 Current Protocols in Cytometry

EtHD-1 (ethidium homodimer 1), 4 mm Dissolve 1 mg of EtHD-1 (Invitrogen/Life Technologies cat. no. E1169) in 275 μl of DMSO. Aliquot and store protected from light and moisture at 20 C. LDS-751 (laser dye styryl-751) working solution, 2 μg/ml Stock solution: Dissolve LDS-751 (Exciton Corp.) at 0.2 mg/ml in methanol. Store up to 1 month at 4 C in the dark. Working solution: Add 0.5 ml stock solution to 50 ml PBS (APPENDIX 2A; 2μg/ml final). Store up to 1 week in the dark at 4 C. Live/Dead Far Red Dead stock solution Prepare a fresh stock solution of Live/Dead Far Red Dead fixable cell stain (Invitrogen/Life Technologies, cat. no. L10120) in DMSO by combining components A and B from the kit according to the manufacturer s instructions. Desiccated dye powder can be stored at 20 C >6 months, while it is recommended that dye in DMSO solution be stored at 20 C and used within 2 weeks of preparation. Sytox AADvanced, 1 mm Add 100 μl of DMSO to one tube of Sytox AADvanced (Invitrogen/Life Technologies, cat. no. S10349) to prepare a 1 mm stock solution. Store protected from light and moisture at 20 C for 1 year avoiding freeze-thaw (manufacturer recommends use immediately after preparation of stock solution). Assessment of Cell Viability 9.2.18 COMMENTARY Background Information Assessments of viability depend on one or both of two cellular properties: (1) the intactness of the cell membrane, and (2) the physiological state of the cell. Dye-exclusion methods are based on the fact that only intact membranes are impermeable to large or charged molecules. Intact membranes also maintain cytoplasmic gradients with respect to the surrounding medium, thus retaining intracellular concentrations of ions and small molecules. This latter property also reflects the physiological state of the cells in that energy is required to maintain gradients. Thus, methods that assay physiological properties of the cell also are dependent upon and indicative of an intact membrane. Probes for membrane integrity Permeability of the cytoplasmic membrane is commonly exploited to mark cells that are moribund or dead. The reagents most often used for this purpose are dyes such as trypan blue or a variety of fluorochromes that will penetrate only damaged permeable membranes of nonviable cells. These are then easily identified visually by the presence of blue color (with trypan blue) in a simple brightfield microscope, or by bright fluorescence seen by fluorescence microscopy or flow cytometry. Fluorescent probes include a wide range of dyes that label DNA of membrane-damaged cells. Tetrabromofluorescein (eosin Y) is a fluorescein derivative that has been used to identify nonviable Candida blastospores (Costantino et al., 1995). In addition, the intracellular penetration of enzymes such as DNase or trypsin can indicate the loss of membrane integrity and thus nonviability (Frankfurt, 1990; Darzynkiewicz et al., 1994; Johnson, 1995). Along the same lines, the penetration of probes for cytoplasmic markers (actin, tubulin, or cytokeratin) has been used to identify cells with damaged plasma membranes (O Brien and Bolton, 1995). The most widely used group of fluorescent probes are those that label nucleic acids (for further discussion of nucleic acid stains, see UNIT 4.3). The most straightforward labeling methods use propidium iodide (PI), 7-amino actinomycin D (7-AAD), or DAPI to identify dead cells, which are hundreds or thousands of times brighter than viable cells. Propidium iodide is in widespread use with many mammalian cell types (Jacobs and Pipho, 1983; Massaro et al., 1989; Coco-Martin et al., 1992; Darzynkiewicz et al., 1994; Stewart and Current Protocols in Cytometry